Repositorium
Journal Article / 2017
Čermák, Tomáš; Curtin, Shaun J.; Gil-Humanes, Javier; Čegan, Radim; Kono, Thomas J. Y.; Konečná, Eva; Belanto, Joseph J.; Starker, Colby G.; Mathre, Jade W.; Greenstein, Rebecca L.; Voytas, Daniel F.
We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
936 |
Voytas, Daniel F. USA |
Hordeum vulgare |
CRISPR/Cas9 MLO |
Resistance against powdery mildew SDN1 |
Basic research Basic research |
937 |
Voytas, Daniel F. USA |
Medicago truncatula |
TALENs Medtr4g020620 |
Phosphate regulation SDN1 |
Basic research Basic research |
938 |
Voytas, Daniel F. USA |
Medicago truncatula |
TALENs Medtr2g013650 |
Phosphate regulation SDN1 |
Basic research Basic research |
939 |
Voytas, Daniel F. USA |
Medicago truncatula |
CRISPR/Cas9 NCR53 |
Phosphate regulation SDN1 |
Basic research Basic research |
940 |
Voytas, Daniel F. USA |
Medicago truncatula |
CRISPR/Cas9 Medtr2g101090 |
Associated with symbiotic nitrogen fixation SDN1 |
Basic research Basic research |
941 |
Voytas, Daniel F. USA |
Nicotiana tabacum |
CRISPR/Cas9 gus:nptII |
No information SDN3 |
Basic research Basic research |
942 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 ARF8A |
Positive regulator of flower development and fruit set SDN1 |
Basic research Basic research |
943 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 Solyc06g074350 |
No information SDN1 |
Basic research Basic research |
944 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 Solyc02g085500 |
No information SDN1 |
Basic research Basic research |
945 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 Solyc02g090730 |
No information SDN1 |
Basic research Basic research |
946 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 Solyc11g071810 |
No information SDN1 |
Basic research Basic research |
947 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 Solyc06g074240 |
No information SDN1 |
Basic research Basic research |
948 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 Solyc02g077390 |
No information SDN1 |
Basic research Basic research |
949 |
Voytas, Daniel F. USA |
Solanum lycopersicum |
CRISPR/Cas9 ANT1 |
No information SDN1 |
Basic research Basic research |
950 |
Voytas, Daniel F. USA |
Triticum aestivum |
CRISPR/Cas9 MLO |
Resistance against powdery mildew SDN1 |
Basic research Basic research |
951 |
Voytas, Daniel F. USA |
Triticum aestivum |
CRISPR/Cas9 Ubiquitin |
No information SDN1 |
Basic research Basic research |
952 |
Voytas, Daniel F. USA |
Triticum aestivum |
CRISPR/Cas9 5-enolpyruvylshikimate-3-phosphate synthase |
No information SDN1 |
Basic research Basic research |
953 |
Voytas, Daniel F. USA |
Triticum aestivum |
CRISPR/Cas9 ubiquitin gene |
No information SDN3 |
Basic research Basic research |